Many normal and pathological functions have been attributed to the oligosaccharide chains on glycoconjugates. With few exceptions, the biosynthesis of these chains and their modifications takes place during trafficking through the Endoplasmic Reticulum-Golgi-Plasmalemma pathway. These glycosylation changes are convenient signposts for the definition of distinctive compartments in these pathways. On the other hand, the pathways can be viewed as kinetic events in the step-wise biosynthesis of biologically important glycoconjugates, that are eventually expressed at the cell surface to serve many biological roles. Each step in the N-linked oligosaccharide (NLO) processing pathway occurs in compartments of the ER- Golgi-PM pathway. Most steps requires a specific nucleotide donor, the corresponding nucleotide transporter, the correct transferase enzyme, and an appropriate endogenous acceptor to all be present at the same time in the same compartment. These complex interactions are not easily studied by pulse-chase analyses, because of the rapid rate of inter-compartmental trafficking. On the other hand, approaches that artificially arrest transport may alter or distort the natural situation. We have demonstrated that all of these steps can be successfully reconstituted in vitro by adding labelled nucleotides to isolated intact membrane preparations from normal tissues and cells. In this setting, inter-compartmental transport is absent, incorporation of label requires intactness of the compartment, and the endogenous products re found to be in the correct topology. This approach also makes it possible to simultaneously study different compartment without fear of mixing them together. The labelled oligosaccharides can then be structurally characterized, giving valuable information regarding the organization and function of the compartments in question. This project focuses on two specific biosynthetic reactions involving N-linked chains: the addition of GlcNAc-1-P units to mannose residues of lysosomal enzymes (an early step), and the addition of 9-0- acetyl esters to the side chains of sialic acid residues on complex-type plasma membrane glycoproteins (a very late step). Studies will be done both in rat liver membrane preparations in permeabilized CHO cells previously engineered to express specific lysosomal enzymes and/or glycosyltransferases. Inter-compartmental transport will be reconstituted by adding back cytosolic factors, and changes in the pre-labelled chains used to elucidate the requirements for trafficking. Finally, these reactions will be compared between the golgi apparatus of normal and malignant hepatocytes, exploring the hypothesis that altered glycosylation in tumor cells is partly explained by a loss of fidelity in Golgi organization. This project directly relates to the primary theme of this program, the study of membrane trafficking, and the role of cytosolic factors in controlling this process. In the long run, the goal is to understand the normal organization of N-linked glycosylation in the Golgi apparatus, its relationship to inter-compartmental trafficking, and the changes that occur in malignant disease.